Smriti Mehra

Abstract
Despite considerable progress in understanding the pathogenesis of Mycobacterium tuberculosis (Mtb), development of new therapeutics and vaccines against it has proven difficult. This is at least in part due to the use of less than optimal models of in-vivo Mtb infection, which has precluded a study of the physiology of the pathogen in niches where it actually persists. C3HeB/FeJ (Kramnik) mice develop human-like lesions when experimentally infected with Mtb and thus make available, a faithful and highly tractable system to study the physiology of the pathogen in-vivo. We compared the transcriptomics of Mtb and various mutants in the DosR (DevR) regulon derived from Kramnik mouse granulomas to those cultured in-vitro. We recently showed that mutant ΔdosS is attenuated in C3HeB/FeJ mice. Aerosol exposure of mice with the mutant mycobacteria resulted in a substantially different and a relatively weaker transcriptional response (< = 20 genes were induced) for the functional category 'Information Pathways' in Mtb:ΔdosR; 'Lipid Metabolism' in Mtb:ΔdosT; 'Virulence, Detoxification, Adaptation' in both Mtb:ΔdosR and Mtb:ΔdosT; and 'PE/PPE' family in all mutant strains compare to wild-type Mtb H37Rv, suggesting that the inability to induce DosR functions to different levels can modulate the interaction of the pathogen with the host. The Mtb genes expressed during growth in C3HeB/FeJ mice appear to reflect adaptation to differential nutrient utilization for survival in mouse lungs. The genes such as glnB, Rv0744c, Rv3281, sdhD/B, mce4A, dctA etc. downregulated in mutant ΔdosS indicate their requirement for bacterial growth and flow of carbon/energy source from host cells. We conclude that genes expressed in Mtb during in-vivo chronic phase of infection in Kramnik mice mainly contribute to growth, cell wall processes, lipid metabolism, and virulence.

Rationale: Hypoxia promotes dormancy by causing physiological changes to actively replicating Mycobacterium tuberculosis (Mtb). DosR controls the response of Mtb to hypoxia. Objectives: To understand its contribution in the persistence of Mtb, we compared the phenotype of various DosR regulon mutants and a complemented strain, to Mtb in macaques, which faithfully model Mtb infection. Methods: We measured clinical and microbiological correlates of infection with Mtb, relative to mutant/complemented strains in the DosR regulon, studied lung pathology and hypoxia and compared immune responses in lung, using transcriptomics and flow-cytometry. Measurements and Main Results: Despite being able to replicate initially, mutants in DosR regulon failed to persist or cause disease. On the contrary, Mtb and a complemented strain were able to establish infection and TB. The attenuation of pathogenesis in animals infected with the mutants coincided with the appearance of a Th1 response and organization of hypoxic lesions wherein Mtb expressed dosR. The lungs of animals infected with the mutants (but not the complemented strain) exhibited early transcriptional signatures of T cell recruitment, activation and proliferation associated with an increase of T cells expressing homing and proliferation markers. Conclusions: Delayed adaptive responses, a hallmark of Mtb infection, not only lead to persistence but also interfere with the development of effective anti-TB vaccines. The DosR regulon therefore modulates both the magnitude and the timing of adaptive immune responses in response to hypoxia in-vivo, resulting in persistent infection. Hence, DosR regulates key aspects of the Mtb life cycle and in limiting lung pathology.

Recently we reported (Mehra et al., 2013), that lung granulomas from Mycobacterium bovis Bacille Calmette-Guérin (BCG)-vaccinated cynomolgus macaques exhibit upon challenge with M. tuberculosis a more balanced expression of α- and β-chemokines, relative to comparable samples from sham-vaccinated animals by comparative transcriptomics. Here, we studied the recruitment of immune cells to blood and lungs in M. tuberculosis-infected macaques as a function of prior BCG-vaccination. Vaccination initially enhanced the levels of both macrophages and lymphocytes in blood. In contrast, significantly more CD4(+) lymphocytes were later recruited to the lungs of sham-vaccinated animals compared with earlier times/BCG vaccinated animals. BCG-vaccination had a short-lived impact on the anti-M. tuberculosis response. M. tuberculosis continued to replicate in the lung even in the wake of increased CD4(+) T cell recruitment to primate lungs, indicating that immune subversive mechanisms are key to its...

Development of a vaccine against pulmonary tuberculosis may require immunization strategies that induce a high frequency of Ag-specific CD4 and CD8 T cells in the lung. The nonhuman primate model is essential for testing such approaches because it has predictive value for how vaccines elicit responses in humans. In this study, we used an aerosol vaccination strategy to administer AERAS-402, a replication-defective recombinant adenovirus (rAd) type 35 expressing Mycobacterium tuberculosis Ags Ag85A, Ag85B, and TB10.4, in bacillus Calmette-Guérin (BCG)-primed or unprimed rhesus macaques. Immunization with BCG generated low purified protein derivative-specific CD4 T cell responses in blood and bronchoalveolar lavage. In contrast, aerosolized AERAS-402 alone or following BCG induced potent and stable Ag85A/b-specific CD4 and CD8 effector T cells in bronchoalveolar lavage that largely produced IFN-γ, as well as TNF and IL-2. Such responses induced by BCG, AERAS-402, or both failed to con...

Mycobacterium tuberculosis (MTB) is a highly successful pathogen because of its ability to persist in human lungs for long periods of time. MTB modulates several aspects of the host immune response. Lymphocyte-activation gene 3 (LAG3) is a protein with a high affinity for the CD4 receptor and is expressed mainly by regulatory T cells with immunomodulatory functions. To understand the function of LAG3 during MTB infection, a nonhuman primate model of tuberculosis, which recapitulates key aspects of natural human infection in rhesus macaques (Macaca mulatta), was used. We show that the expression of LAG3 is highly induced in the lungs and particularly in the granulomatous lesions of macaques experimentally infected with MTB. Furthermore, we show that LAG3 expression is not induced in the lungs and lung granulomas of animals exhibiting latent tuberculosis infection. However, simian immunodeficiency virus-induced reactivation of latent tuberculosis infection results in an increased expression of LAG3 in the lungs. This response is not observed in nonhuman primates infected with non-MTB bacterial pathogens, nor with simian immunodeficiency virus alone. Our data show that LAG3 was expressed primarily on CD4(+) T cells, presumably by regulatory T cells but also by natural killer cells. The expression of LAG3 coincides with high bacterial burdens and changes in the host type 1 helper T-cell response.

The foot processes of astrocytes cover over 60% of the surface of brain microvascular endothelial cells, regulating tight junction integrity. Retraction of astrocyte foot processes has been postulated to be a key mechanism in pathology. Therefore, movement of an astrocyte in response to a proinflammatory cytokine or even limited retraction of processes would result in leaky junctions between endothelial cells. Astrocytes lie at the gateway to the CNS and are instrumental in controlling leukocyte entry. Cultured astrocytes typically have a polygonal morphology until stimulated. We hypothesized that cultured astrocytes which were induced to stellate would have an activated phenotype compared with polygonal cells. We investigated the activation of astrocytes derived from adult macaques to the cytokine TNF-α under resting and stellated conditions by four parameters: morphology, intermediate filament expression, adhesion, and cytokine secretion. Astrocytes were stellated following transient acidification; resulting in increased expression of GFAP and vimentin. Stellation was accompanied by decreased adhesion that could be recovered with proinflammatory cytokine treatment. Surprisingly, there was decreased secretion of proinflammatory cytokines by stellated astrocytes compared with polygonal cells. These results suggest that astrocytes are capable of multiple phenotypes depending on the stimulus and the order stimuli are applied.

We studied the impact of THZ on Mycobacterium tuberculosis (Mtb) by analyzing gene expression profiles after treatment at the minimal inhibitory (1x MIC) or highly inhibitory (4x MIC) concentrations between 1–6 hours. THZ modulated the expression of genes encoding membrane ...

Mycobacterium tuberculosis is one of the most successful pathogens of humankind. During infection, M. tuberculosis must cope with and survive against a variety of different environmental conditions. Sigma factors likely facilitate the modulation of the pathogen's gene expression in response to changes in its extracellular milieu during infection. σH, an alternate sigma factor encoded by the M. tuberculosis genome, is induced by thiol-oxidative stress, heat shock, and phagocytosis. In response to these conditions, σH induces the expression of σB, σE, and the thioredoxin regulon. In order to more effectively characterize the transcriptome controlled by σH, we studied the long-term effects of the induction of σH on global transcription in M. tuberculosis. The M. tuberculosis isogenic mutant of σH (Δ-σH) is more susceptible to diamide stress than wild-type M. tuberculosis. To study the long-term effects of σH induction, we exposed both strains to diamide, rapidly washed it away, and resumed culturing in diamide-free medium (post-diamide stress culturing). Analysis of the effects of σH induction in this experiment revealed a massive temporal programming of the M. tuberculosis transcriptome. Immediately after the induction of σH, genes belonging to the functional categories “virulence/detoxification” and “regulatory proteins” were induced in large numbers. Fewer genes belonging to the “lipid metabolism” category were induced, while a larger number of genes belonging to this category were downregulated. σH caused the induction of the ATP-dependent clp proteolysis regulon, likely mediated by a transcription factor encoded by Rv2745c, several members of the mce1 virulence regulon, and the sulfate acquisition/transport network.

Mycobacterium tuberculosis (Mtb) must counter hypoxia within
granulomas to persist. DosR, in concert with sensor kinases DosS and
DosT, regulates the response to hypoxia. Yet, Mtb lacking functional
DosR colonize the lungs of C57Bl/6 mice, presumably due to the
lack of organized lesions with sufficient hypoxia in that model. We
compared the phenotype of theD-dosR,D-dosS, and D-dosT mutants
to Mtb using C3HeB/FeJ mice, an alternate mouse model where
lesions develop hypoxia. C3HeB/FeJ mice were infected via aerosol.
The progression of infection was analyzed by tissue bacterial burden
and histopathology. A measure of the comparative global immune
responses was also analyzed. Although D-dosR and D-dosT grew
comparably to wild-type Mtb, D-dosS exhibited a significant defect
in bacterial burden and pathology in vivo, accompanied by
ablated proinflammatory response. D-dosS retained the ability to
induce DosR. The D-dosS mutant was also attenuated in murine
macrophages ex vivo, with evidence of reduced expression of the
proinflammatory signature. Our results show that DosS, but not
DosR and DosT, is required by Mtb to survive in C3HeB/FeJ mice.
The attenuation of D-dosS is not due to its inability to induce the
DosR regulon, nor is it a result of the accumulation of hypoxia. That
the in vivo growth restriction of D-dosS could be mimicked ex vivo
suggested sensitivity to macrophage oxidative burst. Anoxic caseous
centers within tuberculosis lesions eventually progress to cavities.
Our results provide greater insight into the molecular mechanisms of
Mtb persistence within host lungs.